Rifamycin R and microorganisms useful in the production of rifamycins

ABSTRACT

Novel rifamycin compounds, designated P, Q, R and U, produced by the fermentation of mutant strains of Streptomyces mediterranei.

CROSS-REFERENCE TO RELATED APPLICATION

This is a division of application Ser. No. 601,559 filed Aug. 4, 1976 now U.S. Pat. No. 4,042,683.

This invention is directed to novel rifamycin compounds which are produced by fermentation of mutant strains of Streptomyces mediterranei in an aqueous nutrient medium under aerobic conditions. These novel rifamycins are hereinafter referred to as rifamycin P, rifamycin Q, rifamycin R and rifamycin U.

It has previously been reported that during fermentation in normal growth media, Streptomyces mediterranei synthesizes a family of antibiotics collectively referred to as the rifamycin complex (P. Sensi, et al., Antibiotics Annual 1959-1960, page 262). Subsequent work revealed that the addition of sodium diethyl barbiturate to the culture medium resulted essentially in the formation of a single fermentation product, rifamycin B (Margalith P. and Pagani H., Applied Microbiology, 9, 325, 1961). According to a further discovery, a mutant strain of Streptomyces mediterranei was found which elaborates essentially rifamycin B only, irrespective of the presence or absence of sodium diethyl barbiturate in the fermentation medium, so that an improved process for making rifamycin B results. The rifamycin B producing strain was identified as Streptomyces Mediterranei ATCC 21789. See U.S. Pat. No. 3,871,965.

The new antibiotic substances which are the object of this invention are produced by mutant strains derived from Streptomyces mediterranei ATCC 13685. The new strains are obtained by treating strain ATCC 13685 with common chemical mutagenic agents such as nitrous acid or nitroso guanidine derivatives or with physical mutagenic agents such as X rays and U.V. radiation. The new mutant strains rather than to the genus Streptomyces mediterranei are assigned to the genus Nocardia in accordance with the proposals by J. E. Thiemann et al., Arch. Mikrobiol., 67, 147-155 (1969).

ISOLATION OF THE MUTATED STRAINS PRODUCING NOVEL RIFAMYCINS

A suspension of spores of Streptomyces mediterranei ATCC 13685 was treated with N-methyl-N'-nitro-N-nitrosoguanidine at 1 mg/ml in a pH 9.0 tris(hydroxymethyl)aminomethane buffer for 60 minutes at 28° C. The mutagen-treated spores were then washed and plated onto Petri dishes containing Bennett agar. After 14 days incubation at 28° C., the surviving colonies were picked off and examined for their ability to inhibit the growth of Bacillus subtilis in the following manner: a disc of agar (diameter 4-8 mm) carrying a single colony was transferred onto a Petri dish containing Penassay agar at pH 7.2 previously seeded at 2% (v/v) with Bacillus subtilis.

Under these conditions rifamycin B is practically inactive and a normal colony producing rifamycin B gives no inhibition of the growth of Bacillus subtilis in the underlying agar, whereas any colony producing a novel rifamycin with antimicrobial activity will cause a clear cut zone of inhibition of growth around the disc of agar. Three strains were isolated in this way and were originally given our internal code D-2, MM18-6 and M-36. Samples of these microorganisms were deposited with the ATCC where they are identified respectively as Nocardia mediterranea ATCC 31064 Nocardia mediterranea ATCC 31065 and Nocardia mediterranea ATCC 31066.

DESCRIPTION OF THE NEW NOCARDIA MEDITERRANEA STRAINS

Table 1 reports the results of the macroscopic and mycroscopic examination of the Nocardia mediterranea strains identified respectively with the ATCC No. 31064, 31065 and 31066, and Table 2 reports the cultural characteristics of the same Nocardia mediterranea strains.

The characteristics of the parent strain Streptomyces mediterranei ATCC 13685 (ME/83) are also reported:

                                      TABLE 1                                      __________________________________________________________________________                Colonies         Aerial mycelium                                                                          Spores                                   __________________________________________________________________________     N. mediterranea                                                                           Colonies conical-crateriform and                                                                Greyish to pinkish,                                                                      Cylindrical                              ATCC 31064 convolute, 2-3 mm. of diameter,                                                                 branched, 0.6-0.8                                                                        (0.8-1 μm × 3.0-5.0μ)                   reddish-brown with ochre yellow                                                                 μm of diameter                                  (D-2)      soluble pigment                                                     N. mediterranea                                                                           Colonies conical-crateriform and                                                                Pink to light orange                                                                     Cylindrical                              ATCC 31065 convulate, 2-3 mm. of diameter,                                                                 1 μ0.6-0.8                                                                            (0.8-μm × 3.0-5.0μ)                     deep orange with deep orange-                                                                   μm of diameter                                  (MM18-6)   yellow soluble pigment                                              N. mediterranea                                                                           Colonies conical-crateriform and                                                                Pink to pink orange                                                                      Cylindrical                              ATCC 31066 convolate, 2-3 mm. of diameter,                                                                 branched, 0.6-0.8                                                                        (0.8-1 μm × 3.0-5.0μ )                  orange brown with amber soluble                                                                 μm of diameter                                  (M-36)     pigment                                                             Streptomyces medi-                                                                        Colonies conical-crateriform and                                                                Pinkish, branched,                                                                       Cylindrical                              terranei ATCC 13685                                                                       convolate, 2-3 mm of diameter,                                                                  0.6-0.8 μm of dia-                                                                    (0.8-1 μm × 3.0-5.0μ )                  orange-yellow with light amber                                                                  meter                                              ME/83      soluble pigment                                                     __________________________________________________________________________

                                      TABLE 2                                      __________________________________________________________________________           N. mediterranea ATCC 31064                                                                   N. mediterranea ATCC 31065                                                                   N. mediterranea ATCC 31066                                                                  Streptomyces mediterranei       Media (D-2)         (MM 18-6)     (M-36)       ATCC 13685                      __________________________________________________________________________                                                    (ME/83)                         Oat meal                                                                             Abundant growth with                                                                         Abundant growth with                                                                         Abundant with smooth sur-                                                                   Fair growth with smooth         agar  slightly wrinkled sur-                                                                       smooth surface, orange.                                                                      face, deep amber.                                                                           surface, yellowish with               face, hazel-brown with                                                                       Traces of pinkish aerial                                                                     Traces of pink aerial                                                                       pinkish reverse. Whitish              orange edges. Pink-beige                                                                     mycelium. Some spores.                                                                       mycelium. Burnt amber                                                                       aerial mycelium with pink             soluble pigment.                                                                             Pink-beige soluble pig-                                                                      soluble pigment.                                                                            tinge. Traces of yellow-                            ment.                      ish soluble pigment.            Yeast Abundant growth with                                                                         Abundant growth with                                                                         Abundant growth with                                                                        Abundant growth with            extract glu-                                                                         very wrinkled surface,                                                                       very wrinkled surface,                                                                       wrinkled surface. Vege-                                                                     rough surface yellowish         cose agar                                                                            amber. Greyish aerial                                                                        brick red. Deep rusty                                                                        tative mycelium, burnt                                                                      to pink. Scanty aerial          (Medium                                                                              mycelium, deep rusty                                                                         soluble pigment.                                                                             amber. Burnt amber so-                                                                      mycelium.                       No. 2 soluble pigment.            luble pigment.                               Shirling                                                                       and                                                                            Gottlieb)                                                                      Emerson's                                                                            Abundant growth with                                                                         Abundant growth with                                                                         Abundant growth with                                                                        Abundant growth with            glucose                                                                              very wrinkled and cru-                                                                       very wrinkled and cru-                                                                       wrinkled surface, burnt                                                                     rough surface, yellowish        agar  sty surface, brick red                                                                       sty surface, brick red,                                                                      amber. Burnt amber solu-                                                                    to pink orange. Pinkish               Amber soluble pigment.                                                                       Amber soluble pigment.                                                                       ble pigment. aerial mycelium. Pale                                                          amber soluble pigment.          Bennett's                                                                            Abundant growth, very                                                                        Abundant growth with                                                                         Abundant growth with                                                                        Good growth, yellowish          agar  wrinkled, reddish brown                                                                      very wrinkled surface,                                                                       wrinkled surface, orange                                                                    turning orange-yellow.                Traces of greyish aerial                                                                     deep orange. Deep yellow                                                                     brown. Amber soluble                                                                        Pinkish aerial mycelium               mycelium. Ochre yellow                                                                       soluble pigment.                                                                             pigment.     Light amber soluble pig-              soluble pigment.                         ment.                           Penassay                                                                             Moderate growth, slight-                                                                     Moderate growth, slight-                                                                     Moderate growth with                                                                        Poor growth                     agar  ly wrinkled light                                                                            ly wrinkled light oran-                                                                      smooth and thin surface,                           orange. No soluble pig-                                                                      ge. No soluble pigment.                                                                      light orange. No soluble                           ment.                       pigment.                                     Yeast Abundant growth                                                                              Abundant growth with                                                                         Abundant growth with cru-                                                                   Abundant growth with                                                           rough                           extract                                                                              very wrinkled, brick red                                                                     very wrinkled surface,                                                                       sty surface, burnt amber                                                                    surface, colorless to           molasse                                                                              Deep rusty soluble pig-                                                                      brick red. Deep rusty                                                                        Burnt amber soluble pig-                                                                    yellowish. Whitish aerial       agar  ment.         soluble pigment.                                                                             ment.        mycelium. Deep amber                                                           solu-                                                                          ble pigment.                    Czapek-                                                                              Moderate growth with                                                                         Good growth with smooth                                                                      Abundant growth with                                                                        Poor growth with thin           Dox   smooth and thin surface                                                                      surface, orange. Light                                                                       smooth surface, salmon.                                                                     surface, colorless to           saccharose                                                                           light orange. Pale pink                                                                      orange aerial mycelium                                                                       Abundant aerial mycelium                                                                    light melon. Traces of          agar  aerial mycelium. Some       pink orange. Traces of                                                                      pinkish white aerial                  spores. Deep lemon yellow   light yellow soluble                                                                        mycelium                              soluble pigment.            pigment.                                     Potato agar                                                                          Scarce growth,light oran-                                                                    No growth     Moderate growth with                                                                        Poor growth with thin                 ge. Light hazel brown       smooth surface. Light                                                                       surface, colorless.                   soluble pigment.            orange. No soluble pig-                                                                     Traces of whitish aerial                                          ment.        mycelium. No soluble                                                           pigment.                        Glucose                                                                              Abundant growth with                                                                         Scarce growth with smooth                                                                    Fair growth, gold.                                                                          Fair growth with smooth         asparagine                                                                           smooth surface orange.                                                                       surface, light orange.                                                                       No soluble pigment.                                                                         and thin surface, light         agar  Light yellow soluble                                                                         No soluble pigment.        orange pink. Some light               pigment.                                 yellow soluble pigment.         Glycerol                                                                       asparagine                                                                           Abundant growth with                                                                         Scarce growth, deep                                                                          Moderate growth with                                                                        Fair growth with smooth         agar  smooth surface deep                                                                          orange.       slightly wrinkled sur-                                                                      and thin surface. Light         (medium                                                                              orange. Traces of                                                                            No soluble pigment.                                                                          face, deep orange. Traces                                                                   orange pink. Some light         n° 5 Shir-                                                                    yellowish soluble pig-      of yellow soluble pig-                                                                      yellow soluble pigment.         ling and                                                                             ment.                       ment.                                        Gottlieb)                                                                      Nutrient                                                                             Moderate growth with                                                                         Moderate growth with                                                                         Fair growth with thin                                                                       Moderate growth with                                                           smooth                          agar  thin and smooth sur-                                                                         wrinkled and thin sur-                                                                       and smooth surface,                                                                         surface, melon to orange.             face, pale orange.                                                                           face, pale orange.                                                                           orange. No soluble                                                                          Pinkish white aerial my-              No soluble pigment.                                                                          No soluble pigment.                                                                          pigment.     celium.                         Pridham's                                                                            Moderate growth with                                                                         Abundant growth with                                                                         Abundant growth, slight-                                                                    Moderate growth with            agar  smooth and thin surface,                                                                     smooth surface, brick                                                                        ly crusty, amber brown.                                                                     smooth surface, colorless             amber. Light pinkish-                                                                        red. Pink aerial myce-                                                                       Traces of pinkish aerial                                                                    with lobster red spots.               orange aerial mycelium.                                                                      lium. Yellow soluble                                                                         mycelium.    Pink aerial mycelium.                 Pinkish soluble pigment.                                                                     pigment.      Amber brown soluble pig-                                                       ment.                                        Starch                                                                               Moderate growth with                                                                         Moderate growth with                                                                         Abundant growth with                                                                        Poor growth, colorless to       agar  smooth surface orange.                                                                       smooth surface, deep                                                                         smooth surface, orange.                                                                     light orange pink.              (medium                                                                              Abundant aerial myce-                                                                        orange.       Traces of pinkish aerial                                                                    Scarce white aerial myce-       n° 4 Shir-                                                                    lium, pink. Abundant                                                                         Traces of straw soluble                                                                      mycelium. Light yellow                                                                      lium. Starch hydrolysis:        ling and                                                                             production of spores.                                                                        pigment. Starch hydro-                                                                       soluble pigment. Starch                                                                     doubtful.                       Gottlieb)                                                                            Yellow soluble pigment.                                                                      lysis: negative.                                                                             hydrolysis: negative.                              Starch hydrolysis: nega-                                                       tive.                                                                    Dextrose                                                                             Abundant growth with                                                                         Moderate growth, wrin-                                                                       Abundant growth with                                                                        Abundant growth pink            trip- wrinkled surface melon.                                                                      kled, orange. Orange                                                                         wrinkled surface, rose-                                                                     orange. Pinkish aerial          tone agar                                                                            Orange melon soluble                                                                         yellow soluble pigment.                                                                      melon. Rose melon solu-                                                                     mycelium. Light golden                pigment.                    ble pigment. yellow soluble pigment.         Hickey's                                                                             Moderate growth with                                                                         Abundant growth with                                                                         Moderate growth with                                                                        Moderate growth, light          and   smooth and thin surface,                                                                     smooth surface, rose                                                                         slightly wrinkled sur-                                                                      pinkish orange. Some            Tresner's                                                                            colorless. Whitish aerial                                                                    melon. Light beige-rose                                                                      face, rose-melon. Light                                                                     pinkish aerial mycelium.        cobalt agar                                                                          mycelium. Beige soluble                                                                      soluble pigment.                                                                             beige soluble pigment.                                                                      Yellowish soluble                                                              pigment.                              pigment.                                                                 Tyrosine                                                                             Abundant growth, very                                                                        Abundant growth, very                                                                        Abundant growth with                                                                        Poor growth                     agar  wrinkled and crusty                                                                          wrinkled and crusty.                                                                         crusty surface, deep                         (Medium                                                                              brick red. Deep yellow                                                                       Deep orange. Pinkish-                                                                        burnt orange. Yellow                         n° 7 Shir-                                                                    soluble pigment. Tyrosi-                                                                     tan soluble pigment.                                                                         ochre soluble pigment.                       ling and                                                                             ne reaction: positive                                                                        Tyrosine reaction:                                                                           Tyrosine reaction:                           Gottlieb)                                                                            (good).       strongly positive.                                                                           strongly positive.                           Ca-malate                                                                            Scarce growth with                                                                           Scarce growth, light                                                                         Poor growth, light                                                                          Fair growth colorless           agar  smooth and thin surface,                                                                     orange. Hydrolysis of                                                                        orange. No soluble pig-                                                                     Whitish pink aerial                   light orange yellow.                                                                         Ca-malate: negative.                                                                         ment. Ca-malate dige-                                                                       mycelium. Partial dige-               Chrome lemon soluble        stion: negative.                                                                            stion of Ca-malate.                   pigment near the growth                                                        and coral pink in the                                                          medium. Hydrolysis: po-                                                        sitive.                                                                  Egg   Abundant growth with                                                                         Abundant growth with                                                                         Good growth with smooth                                                                     Fair growth, pink.              albumin                                                                              smooth surface, light                                                                        smooth surface, orange.                                                                      surface orange-yellow.                       agar  orange. Light yellow                                                                         No soluble pigment.                                                                          Light yellow soluble pig-                          soluble pigment.            ment.                                        Peptone                                                                              Abundant growth, slight-                                                                     Abundant growth, wrin-                                                                       Abundant growth with                         glucose                                                                              ly wrinkled, deep oran-                                                                      kled, deep orange.                                                                           wrinkled surface, burnt                      agar  ge. Pinkish aerial myce-                                                                     Gold yellow soluble                                                                          amber. Burnt amber so-                             lium. Yellow soluble pig-                                                                    pigment.      luble pigment.                                     ment.                                                                    Gelatine                                                                             hydrolysis: positive                                                                         hydrolysis: positive                                                                         hydrolysis: positive                                                                        hydrolysis: positive            Nitrate                                                                              reduction: negative                                                                          reduction: negative                                                                          reduction: negative                                                                         reduction: negative             broth                                                                          Litmus                                                                               no coagulation                                                                               no coagulation                                                                               no coagulation                                                                              no coagulation                  milk  no peptonization                                                                             no peptonization                                                                             no peptonization                                                                            no peptonization                Peptone                                                                              Moderate growth with                                                                         Moderate growth with                                                                         Poor growth with                             yeast smooth surface, pale                                                                         smooth surface, pale                                                                         smooth surface, color-                       extract                                                                              orange. H.sub.2 S production:                                                                orange. H.sub.2 S production:                                                                less. No soluble pig-                        iron agar                                                                            negative      negative.     ment. H.sub.2 S production:                  (Medium                           negative.                                    n° 6 Shir-                                                              ling and                                                                       Gottlieb)                                                                      Potato plug                                                                          Scarce growth, orange.                                                                       Very scarce growth,                                                                          Poor growth, light or-                                                                      Poor growth, colorless.                             orange.       ange.                                        Loeffler                                                                             No growth     No growth     No growth                                    blood                                                                          serum                                                                          H.sub.2 O +                                                                          Scarce growth colorless.                                                                     Scarce growth, colorless.                                                                    Scarce growth, colorless                     agar  Traces of white aerial                                                                       Traces of white aerial                                                                       Traces of white aerial                       Difco 2%                                                                             mycelium. Abundant pro-                                                                      mycelium. Abundant pro-                                                                      mycelium. Abundant pro-                            duction of spores.                                                                           duction of spores.                                                                           duction of spores.                           Skim milk                                                                            Abundant growth with                                                                         Good growth, slightly                                                                        Abundant growth with                         agar  smooth surface, orange.                                                                      wrinkled, orange with                                                                        wrinkled surface, oran-                            Cadmium yellow soluble                                                                       brown shadow. ge. Gold brown soluble                             pigment. Hydrolysis of                                                                       Hazel brown soluble                                                                          pigment. Casein hydroly-                           casein: good. pigment. Hydrolysis of                                                                       sis: strongly positive.                                          casein: good.                                              __________________________________________________________________________      Shirling and Gottlieb: Methods for characterization of Streptomyces            species, Intern. J. Syst.Bact., 16,313-338 (1966).                       

The following table 3 reports the physiological characteristics of the new Nocardia Strains compared with those of the parent strain.

                                      TABLE 3                                      __________________________________________________________________________             N.mediterranea                                                                          N.mediterranea                                                                          N.mediterranea                                                                          Streptomyces mediterranei                           (D-2)    (MM 18-6)                                                                               (M-36)   (ME/83)                                     __________________________________________________________________________     Strach hydro-                                                                          -        -        -        ±                                        lysis                                                                          Tyrosin hydro-                                                                         ++       +++      +++      -                                           lysis                                                                          Casein hydro-                                                                          ++       ++       +++                                                  lysis                                                                          Ca-malate                                                                              ++       -        -        +                                           hydrolysis                                                                     Nitrate re-                                                                            -        -        -        -                                           duction                                                                        Litmus milk                                                                            no coagulation                                                                          no coagulation                                                                          no coagulation                                                                          no coagulation                                      no peptonization                                                                        no peptonization                                                                        no peptonization                                                                        no peptonization                            Gelatin ++       ++       ++       +                                           liquefaction                                                                   __________________________________________________________________________

Following Table 4 reports the utilization of carbon sources examined according to the method of Pridham and Gottlieb (J.Bact. 56, 107, 1948).

                                      TABLE 4                                      __________________________________________________________________________            N.mediterranea                                                                         N.mediterranea                                                                         N.mediterranea                                                                         Streptomyces mediterranei                              (D-2)   (MM 18-6)                                                                              (M-36)  (ME/83)                                         __________________________________________________________________________     Arabinose                                                                             +       +++     ++      +++                                             Xylose +       +       ++      ++                                              Glucose                                                                                ++     +++     ++      ++                                              Mannose                                                                               +       ++      ++      ++                                              Fructose                                                                               ++     +++     ++      ++                                              Lactose                                                                               +       +++     ++      ++                                              Sucrose                                                                               +       ±    ++      ++                                              Inositol                                                                              +       +       + +     ++                                              Rhamnose                                                                              +       +       ++      ++                                              Raffinose                                                                             -       -       -       -                                               Salicin                                                                               ±    +       +       +                                               Mannitol                                                                               ++     +++     ++      ++                                              Glicine                                                                               +       ±    +       -                                               Sorbitol                                                                              -       -       -       -                                               Ma-succinate                                                                          -       -       -       +                                               Na-citrate                                                                            -       -       -       -                                               Na-acetate                                                                            -       -       -       -                                               Inulin ±    +       -       -                                               Dulcitol                                                                              -       -       -       -                                               Cellulose                                                                             -       -       -       -                                               Maltose                                                                                ++     +++     ++      +                                               Galactose                                                                             +       ++      ++      +++                                             Glicerol                                                                               ++     +++     ++      +++                                             __________________________________________________________________________

FERMENTATION

The procedure for the fermentation essentially consists in cultivating one of said mutants of Streptomyces mediterranei in a nutrient medium containing assimilable carbon and nitrogen sources and essential mineral salts, until a substantial antibiotic activity is imparted to said medium and in extracting the rifamycins from the medium. More particularly these mutants are cultivated under stirred and aerated submerged conditions at a temperature ranging from 25° to 37° C., and preferably at 28° C. As the sources of carbon the following carbohydrates and carbon derivatives can be used: glucose, galactose, lactose, sucrose, maltose, glycerol, mannitol, etc. Useful nitrogen sources are for instance amino-acids and their mixtures, peptides, proteins and their hydrolisates, as peptone, yeast extract, soybean meal, corn steep liquor, fish soluble, meet extracts, aqueous fractions from cereal seeds. The fermentation can be carried out for 180-220 hours. The starting pH, generally adjusted at about 6.4-6.6 increases at the end of the fermentation to 7.0-8.5. Generally the best results are observed at the 200 hours of the fermentation. At the end of the fermentation the rifamycins can be isolated by the following procedure. The fermentation medium is filtered at the final pH of 7.0-8.5. The filtrate is quickly acidified, preferably to a pH lower than about 5, to ensure the best stability to the antibiotic substance. The activity is extracted with water immiscible solvents such as chloroform, butanol, ethyl, propyl, butyl or amyl acetate. The ratio between the volume of the medium and that of the solvent changes dependently on the choosen solvent: generally a ratio ranging from 2:1 to 10:1 is used.

The mycelium still retains a microbiological activity, which is extracted from the mycelium by means of a water immiscible solvent, and then combined with the organic phase, already containing most of the rifamycins. Alternatively, the extraction of the activity from the mycelium can be effected by means of a solvent miscible with water such as acetone. In this case, the liquid is filtered, the acetone is evaporated in vacuo, the rifamycins are extracted with a water immiscible solvent and the procedure is carried on as said above.

Once most of the antibiotic activity has been transferred into the solvent, this is distilled in vacuo to dryness, preferably at a temperature lower than 30° C.

PURIFICATION

The crude extract of rifamycins can be purified chromatographically on a column of silica-gel. Prior to chromatography it is convenient to dissolve the crude extract in a phosphate buffer pH 7.0-8.0 and to treat with a mild oxidizing agent. The buffer solution is then extracted with a water-immiscible solvent, this organic extract contains a new rifamycin denominated rifamycin P. The extracted buffer solution is acidified to pH 2-4 and then again extracted with a water immiscible solvent. This organic extract contains three new compounds of the rifamycin family denominated rifamycin P, Q and U.

The further purification of rifamycin R is effected by chromatography on a column of suitable adsorbing material such as silica-gel and eluting with an appropriate mixture of organic solvents.

The second organic extract containing rifamycins P, Q and U is purified in a similar way to that described for rifamycin R.

The following examples are given for the purpose of better illutrating the subject of the invention.

EXAMPLE 1

The mutant strains identified as Nocardia mediterranea ATCC 31064 is propagated for 6-8 days on Bennett's agar and incubated at 28° C.

With the culture obtained from the agar slant, two 500 ml. Erlenmeyer flasks are inoculated under sterile conditions. The flasks contain 100 ml. of the vegetative medium of the following composition:

    ______________________________________                                         Beef extract           5      g.                                               Yeast extract          5      g.                                               Peptone                5      g.                                               Casein hydrolyzate     3      g.                                               Glucose                20     g.                                               NaCl                   1.5    g.                                               H.sub.2 O to           1      liter.                                           ______________________________________                                    

The pH is adjusted to 7.3 with NaOH.

The flasks so inoculated are placed on an alternative shaker at 28° C. for 72 hours. The content of the two Erlenmeyer flasks is used as inoculum by pouring it in a 10 liters prefermenter, containing 4 liters of the above mentioned vegetative medium. The incubation is carried out at 28° C. with an agitation of 300 r.p.m. and 1 v/v/m aeration. After 48 hours of growth a volume of 7-10% of packed cells is obtained. In the next stage a 10 liter glass fermenter containing 4 liters of the hereinafter mentioned fermentation medium is used:

    ______________________________________                                         Peanut flour          25     g.                                                Soybean flour         5      g.                                                (NH.sub.4).sub.2 SO.sub.4                                                                            9.5    g.                                                MgSO.sub.4 . 7H.sub.2 O                                                                              0.85   g.                                                Glucose               95     g.                                                Glycerol              40     g.                                                KH.sub.2 PO.sub.4     1      g.                                                Propylene glycol      5      g.                                                CaCO.sub.3            8.5    g.                                                Na diethylbarbiturate 1.7    g.                                                CuSO.sub.4 . 5H.sub.2 O                                                                              2.8    mg.                                               FeSO.sub.4 . 7H.sub.2 O                                                                              8.5    mg.                                               ZnSO.sub.4 . 7H.sub.2 O                                                                              42.5   mg.                                               MnSO.sub.4 . 4H.sub.2 O                                                                              3.4    mg.                                               CaCl.sub.2 . 6H.sub.2 O                                                                              1.7    mg.                                               (NH.sub.4).sub.6 Mo.sub.7 O.sub.24 . 4H.sub.2 O                                                      0.85   mg.                                               H.sub.2 O to          1      liter                                             ______________________________________                                    

The pH is adjusted to 7.8 with NaOH. Sterilization for 60 minutes at 120° C. After sterilization the pH is 6.4. An amount of the prefermenter content equal to 5% of the fermenter content is used as inoculum.

The fermentation is carried out at 28° C. with a 750 r.p.m. agitation and aerating at a rate of 1 v/v/m. Silicone A is used as antifoam. The culture broth turns to a characteristic red-brown color during the fermentation. After 200 hours of growth a volume of packed cells is obtained. The pH of the broth is 7.5 and at this moment the broth is harvested.

EXAMPLE 2

A culture of Nocardia mediterranea ATCC 31064 obtained as disclosed in example 1, is prepared in a flask, under stirring, as described in example 1. For the preculture it is poured into a 10 liters glass fermenter, containing 4 liters of the following medium:

    ______________________________________                                         Glucose              5      g.                                                 Peanut flour         7.5    g.                                                 CaCO.sub.3           1.65   g.                                                 MgSO.sub.4 . 7H.sub.2 O                                                                             0.33   g.                                                 KH.sub.2 PO.sub.4    0.33   g.                                                 FeSO.sub.4 . 7H.sub.2 O                                                                             3.3    mg.                                                ZnSO.sub.4 . 7H.sub.2 O                                                                             16.5   mg.                                                MnSO.sub.4 . 4H.sub.2 O                                                                             1.3    mg.                                                H.sub.2 O to         1      liter                                              ______________________________________                                    

The pH is adjusted to 7.5. Sterilization 50 minutes at 120° C. After sterilization the pH value is 6.4. After 48 hours of growth the volume of packed cells is 6-8% of total volume. An inoculum equal to 10% is used for a 20 liters glass fermenter, containing 10 liters of the following fermentation medium:

    ______________________________________                                         Corn steep liquor    20      g.                                                Soybean flour        15      g.                                                (NH.sub.4).sub.2 SO.sub.4                                                                           6       g.                                                MgSO.sub.4 . 7H.sub.2 O                                                                             0.85    g.                                                Glucose              100     g.                                                KH.sub.2 PO.sub.4    1       g.                                                CaCO.sub.3           6       g.                                                FeSO.sub.4 . 7H.sub.2 O                                                                             8.5     mg.                                               ZnSO.sub.4 . 7H.sub.2 O                                                                             42.5    mg.                                               MnSO.sub.4 . 4H.sub.2 O                                                                             3.4     mg.                                               CuSO.sub.4 . 5H.sub.2 O                                                                             2.8     mg.                                               CoCl.sub.2 . 6H.sub.2 O                                                                             1.7     mg.                                               H.sub.2 O to         1       liter                                             ______________________________________                                    

The pH is adjusted to 7.8 with NaOH. Sterilization for 50 minutes at 120° C. After sterilization the pH is 6.4. The fermentation is carried out at 28° C. for 200 hours. The pH of the fermentation broth at the harvest is 7.5.

The fermentation broths obtained in examples 1 and 2 are purified in the following way. The mycelium is removed by filtration and discarded, the filtrate is adjusted to pH 2.0 with 10% (v/v) hydrochloric acid and extracted three times with an equal volume of ethyl acetate. This organic extract is concentrated to dryness under vacuum at 35° C. and the residue (4 g. from example 1 and 11 g. from example 2) is dissolved in 0.05 M sodium phosphate buffer pH 7.5 and sodium nitrite added to give a final concentration of 0.2% (w/v). After stirring for 30 min. at room temperature the buffer solution is extracted three times with an equal volume of ethyl acetate. The combined organic extracts are concentrated to dryness under vacuum at 35° C. (1^(st) ethyl acetate extract). The exhausted buffer solution is adjusted to pH 2.0 with 10% hydrochloric acid and extracted with an equal volume of ethyl acetate three times. The combined organic extracts are concentrated to dryness under vacuum at 35° C. (2^(nd) ethyl acetate extract).

The dry powder from the 1^(st) ethyl acetate extract (1.4 g example 1 and 4.2 g. example 2) is dissolved in chloroform and chromatographed on a column of silica-gel (70-230 Mesh ASTM) using chloroform containing 2% (v/v) methyl alcohol as an eluent. Rifamycin R is the first major product to elute from the column recognizable by its orange-brown colour.

On thin layer chromatography using silica-gel plates (merck 60 F₂₅₄) with chloroform: methyl alcohol as solvent system (95:5) rifamycin R has an Rf of 0.59. The fractions containing rifamycin R are combined, taken to dryness under vacuum at 35° C., redissolved in ethyl acetate which is then washed with 0.01 N hydrochloric acid and finally with water. The organic extract is concentrated to a small volume and rifamycin R crystallizes from solution at 4° C. (obtained 600 mg. from example 1 and 1.6 g. from example 2). The second ethyl acetate extract is purified by column chromatography on silica-gel in a similar way to rifamycin R. The dry residue (2.4 g. from example 1 and 4.2 g. from example 2) is dissolved in chloroform and applied to the silica gel.

The column is eluted with a mixture of chloroform and methyl alcohol (98:2). The first compound to emerge is rifamycin U, then rifamycin P and finally rifamycin Q. All three of these compounds have a yellow orange colour in the eluent solution and can be identified on the bases of their mobility on thin layer chromatography. On silica gel plates (Merck 60 F₂₅₄) using chloroform: methanol (95:5) as solvent system, the Rf values are the following:

    ______________________________________                                                rifamycin U   Rf = 0.63                                                        rifamycin P   Rf = 0.57                                                        rifamycin Q   Rf = 0.32                                                 ______________________________________                                    

The appropriate fractions containing rifamycins U, P and Q are combined taken to dryness under vacuum and crystallized from ethyl acetate. From the first example are obtained rifamycin U 80 mg., rifamycin P 400 mg., rifamycin Q 280 mg: from the second example rifamycin U 190 mg., rifamycin P 900 mg., rifamycin Q 750 mg.

By following essentially the same procedure of the above examples but using respectively Nocardia mediterranea ATCC 31065 or Nocardia mediterranea ATCC 31066 as the producing strains, yields of the same order as above are obtained.

PHYSICO-CHEMICAL CHARACTERISTICS OF THE NOVEL RIFAMYCINS Rifamycin P

(1) Elemental analysis (%): Found, C=60.6; H=6.2; N=3.8; O=25.2; S=4.1.

(2) U.V. and visible absorption bands:

The compound shows the following values:

    ______________________________________                                         methanol            0.1 N HCl                                                  ______________________________________                                         λ.sub.max(mμ)                                                                    E.sub.1.sup.1% cm                                                                          λ.sub.max (mμ)                                                                  E.sub.1.sup.1% cm                           406         197         416        183                                         350         (shoulder)  300        319                                         297         344         225        521                                         257         423                                                                224         550                                                                ______________________________________                                    

The complete figure of the spectrum is given in FIG. 1. The spectrum was recorded with Perkin Elmer Spectracord 4000 A instrument.

(3) Infrared Spectrum: The most significant absorption peaks in Nujol occur at the following frequencies (cm⁻¹): 3700-3150 (m,br); 3100 (w); 3060-2800 (vs); 1465 (s); 1380 (b): Nujol; 1722 (m); 1645 (m,br); 1580 (m); 1510 (m); 1325 (m); 1250 (s br); 1160 (m); 1130 (w); 1070 (m, br) 1030 (w); 982 (m); 960 (m); 925 (w); 890 (m); 818 (w); 770 (w); 735 (w).

A complete figure of the I.R. spectrum is given in FIG. 2. The spectrum was recorded with a Perkin Elmer Mod. 421 instrument.

(4) Mass spectrum: The mass spectrum obtained at 70 eV shows the molecular ion peak M⁺ at the following m/e value: 738. The spectrum was recorded with an Hitachi Perkin Elmer RMU-6L instrument.

(5) Nuclear Magentic Resonance spectrum: The complete figure of N.M.R. spectrum at 100 MH_(z) in CDCl₃ is given in FIG. 3. This compound does not show the characteristic polarographic behavior of the rifamycins possessing a chromophoric moiety with quinonic structure.

Rifamycin O

(1) Elemental analysis: Found, C=60.7; H=6.3; N=3.60; O=25.3; S=4.2

(2) U.V. and visible absorption bands:

The compound shows the following values in methanol:

    ______________________________________                                         λ.sub.max (mμ)                                                                            E.sub.1.sup.1% cm                                         406                  178                                                       350                  (shoulder)                                                297                  305                                                       257                  405                                                       224                  518                                                       ______________________________________                                    

(3) Infrared Spectrum: The most significant absorption peaks in Nujol occur at the following frequencies (cm⁻¹): 3700-3300 (s,br); 3300-3080 (m br); 3040-2780 (vs); 1460 (s); 1378 (s): Nujol; 1740 (m); 1700 (m); 1650 (s, br); 1605 (s, br); 1555 (s); 1510 (m, br); 1315 (w); 1275 (m); 1240 (m); 1220 (m); 1160 (m); 1090 (m); 1050 (m,br); 1020 (w); 970 (m); 945 (w); 910 (m); 808 (m); 765 (w); 720 (w).

A complete figure of the I.R. spectrum is given in FIG. 4.

(4) Nuclear Magnetic Resonance Spectrum: The complete figure of the N.M.R. spectrum at 60 MH_(z) in CDCl₃ is given in FIG. 7.

Rifamycin R

(1) Elemental analysis: Found: C=62.0; H=6.4; N=2.2; O=29.6.

(2) U.V. and visible absorption bands:

The compound shows the following values in which 0.1 N HCl in methanol:

    ______________________________________                                                λ.sub.max (mμ)                                                                     E.sub.1.sup.1% cm                                                219           444                                                              281           415                                                              340           114                                                              410           72                                                        ______________________________________                                    

(3) Infrared Spectrum: The most significant absorption peaks in Nujol occur at the following frequencies (cm⁻¹): 3700-3100 (s br); 3040-2780 (vs); 1465 (s); 1380 (s): Nujol; 1745 (s); 1710 (m); 1640 (s); 1600 (s); 1505 (s); 1415 (m); 1325 (s); 1260 (s, br); 1220-1130 (s, br); 1120 (w); 1075 (s); 1020 (w); 975 (s); 950 (w); 920 (w); 888 (m); 823 (m); 785 (w, br); 735 (w, br); 650 (w, br).

A complete figure of the I.R. spectrum is given in FIG. 5.

(4) Mass spectrum: The mass spectrum obtained at 70 eV shows the molecular ion peak M⁺ at the following m/e value: 711.

(5) Nuclear Magnetic Resonance spectrum: The complete figure of the N.M.R. spectrum at 60 MH_(z) in CDCl₃ is given in FIG. 6.

The compound shows the characteristic polorographic behavior of the rifamycins possessing a chromophoric moiety with quinonic structure.

The above data support the following structure for rifamycin R. ##STR1##

Rifamycin U

(1) U.V. and visible absorption bands:

The compound shows the following values in methanol:

    ______________________________________                                         λ.sub.max (mμ)                                                                            E.sub.1.sup.1% cm                                         412                  163                                                       353                  (shoulder)                                                299                  265                                                       258                  389                                                       220                  451                                                       ______________________________________                                    

(2) Infrared spectrum: The most significant absorption peaks in Nujol occur the following frequencies (cm⁻¹): 3700-3100 (m,br); 3060 (vw); 3040-2740 (vs), 2720 (vw), 1460 (s), 1378 (m): Nujol; 1750-1705 (m,br); 1680-1620 (m,br); 1600 (s); 1558 (s); 1490 (w, shoulder); 1308 (w); 1270 (w, shoulder); 1245 (m, shoulder); 1225 (m); 1160 (m); 1120 (w); 1090 (vw); 1070 (w); 1030 (vw); 1020 (vw); 1000 (vw); 975 (w); 960 (w, shoulder); 905 (m); 850 (m); 802 (w); 800 (vw, shoulder); 777 (vw); 760 (vw); 720 (vw); 685 (vw); 670 (vw).

BIOLOGICAL ACTIVITY OF THE COMPOUNDS

The in vitro activity spectrum of rifamycins P, Q and R is reported in the following table:

    __________________________________________________________________________                        Minimal inhibition concentration (μg/ml)                 Strains            Rifamycin P                                                                           Rifamycin Q                                                                           Rifamycin R                                                                           Rifamycin M                            __________________________________________________________________________     Staphylococcus aureus ATCC 6538                                                                   0.00235                                                                               0.039  0.019  0.05-0.1                               Staphylococcus aureus ATCC 6538                                                                   0.00235                                                                               0.078  0.0047 --                                     (20% bovine serum)                                                             Staphylococcus aureus Tour                                                                        0.0035 0.06   0.00235                                                                               --                                     Staphylococcus aureus Tour                                                     rifampicin-resistant                                                                              25-50  >100   100    --                                     Streptococcus hemolyticus C 203                                                                   0.0047 0.019  0.00312                                                                               --                                     Streptococcus faecalis ATCC 10541                                                                 0.0035 0.312  0.19   --                                     Diplococcus pneumoniae UC 41                                                                      0.0047 0.039  0.00625                                                                               --                                     Proteus vulgaris X 19 N ATCC 881                                                                  0.78   12.5   12.5   --                                     Escherichia coli ATCC 10536                                                                       3.12   12.5   50     >50                                    Klebsiella pneumoniaeATCC 10031                                                                   12.5   50     --     --                                     Pseudomonas aeruginosaATCC 10145                                                                  12.5   50     100    --                                     Mycobacterium tub. H37Rv ATCC 9360                                                                1      2.5    0.1    2.5                                    __________________________________________________________________________      --: Activity not determined?                                             

The novel compounds are active also in the laboratory animals injected with Staphylococcus aureus, Escherichia coli. The following table reports the results of representative experiments on mice in comparison with a known natural rifamycin, i.e. rifamycin SV.

    ______________________________________                                                               ED.sub.50 mg/kg.                                         Compound   Infecting Strain s.c.    o.s.                                       ______________________________________                                         rifamycin P                                                                               Staphylococcus aureus                                                                           0.3     0.8                                        rifamycin P                                                                               Escherichia coli 40      --                                         rifamycin Q                                                                               Staphylococcus aureus                                                                            8      --                                         rifamycin SV                                                                              Staphylococcus aureus                                                                           17      74                                         ______________________________________                                    

Rifamycin P which results to be the most active natural rifamycin so far isolated has also low toxicity since its LD₅₀ value in mice is higher than 500 mg/kg. i.p. 

We claim:
 1. A biologically pure culture of the microorgansim Nocardia mediterranea, having the identifying characteristics of ATCC 31064, said culture being capable of producing an antibiotic selected from the group consisting of rifamycin P, Q, R, and U in a recoverable quantity upon aerobic fermentation in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen, and inorganic minerals.
 2. A biologically pure culture of the microorganism Nocardia mediterranea, having the identifying characteristics of ATCC 31065, said culture being capable of producing an antibiotic selected from the group consisting of rifamycin P, Q, R, and U in a recoverable quantity upon aerobic fermentation in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen, and inorganic minerals.
 3. A biologically pure culture of the microorganism Nocardia mediterranea, having the identifying characteristics of ATCC 31066, said culture being capable of producing an antibiotic selected from the group consisting of rifamycin P, Q, R, and U in a recoverable quantity upon aerobic fermentation in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen, and inorganic minerals.
 4. A rifamycin compound defined as rifamycin R having the following formula: ##STR2## 